Concentration-dependent Interaction of Metformin and Caffeine in HCC1937 Triple-Negative Breast Cancer Cells: An in vitro Study

Abstract

Background: Triple-negative breast cancer (TNBC) remains a clinically challenging breast cancer subtype, motivating the investigation of repurposed compounds and rational combinations. Metformin and caffeine affect distinct cellular processes, but their quantitative interaction in BRCA1-altered TNBC cells is insufficiently characterised. Methods: HCC1937 BRCA1-altered TNBC cells were exposed to metformin and caffeine individually or in sequential combinations. MTT reduction after 48 h was used as a proxy for metabolically active cell mass. Concentration-response relationships were fitted using a four-parameter logistic model, and drug interactions were evaluated using the Bliss independence model and expressed as excess-over-Bliss (ΔBliss) values. Results: Caffeine and metformin reduced MTT signal in a concentration-dependent manner. The estimated caffeine IC₅₀ was 8.6 mM. Metformin did not reach 50% inhibition within the tested 0-20 mM range; its estimated IC₅₀ was approximately 22.0 mM and therefore represents an extrapolated value. In the sequence using 5 mM metformin followed by caffeine, the apparent caffeine IC₅₀ shifted to approximately 1.8 mM. The combination of 0.5 mM caffeine with 5 mM metformin produced 35% observed inhibition versus 28% expected under Bliss independence (ΔBliss = +0.07), whereas other tested pairs were consistent with Bliss independence or showed below-Bliss responses. Conclusion: Metformin and caffeine produced concentration-dependent reductions in MTT-derived metabolic activity in HCC1937 cells, and their interaction varied across the tested concentrations and treatment sequences. The findings support further evaluation using broader concentration matrices, replicate-level interaction statistics, orthogonal viability or cell-death assays, and exposure conditions with greater translational relevance.